Wolfgang R. Deppert

Critical evaluation of human endometrial explants as an ex vivo model system: a molecular approach

The human endometrium is unique among adult tissues. Its functions are modulated by numerous hormones and mediators. The aim of this study was to evaluate the suitability of human endometrial explants for studying functional effects of chemicals and drugs on gene expression biomarkers. Endometrial tissues were obtained by aspiration curettage and cultivated for up to 24 h. Relative mRNA concentrations were determined by reverse transcription quantitative real-time PCR. Viability was assessed by light microscopy, lactate dehydrogenase assay and scanning electron microscopy. It was acceptable after 6 h of culture but reduced after 24 h. Culture-induced alterations of mRNA levels were found for progesterone receptor, estrogen receptor(α), leukemia inhibitory factor and cyclooxygenase-2 in tissues from all cycle stages. The suitability of the model to detect chemical effects was demonstrated by the down-regulation of cyclooxygenase-2 mRNA by chlormadinone acetate in proliferative and secretory endometrium. The model is mainly restricted by interindividual variations and varying tissue quality. An advantage is the preservation of tissue composition. We conclude that human endometrial explants are a complex model due to limited viability, difficult standardization and intrinsic alterations during culture. Experiments with this model should be performed over a limited time period under strictly controlled conditions.

Purification of Adenylate Kinase from Green Leaves ofBarley, Maize and Chenopodium rubrum L.

Summary Adenylate kinase (ATP: AMP phosphotransferase, E.C. 2.7.4.3) plays an important role in the regulationof energy metabolism in all living systems. The enzyme has been purified and crystallized from a variety of animals and microorganisms, while purification from plant tissues was described only for leaves from the C 4 -plant maize. Here, we present an efficient purification procedure for adenylate kinase derived from green leaves of the C 3 -plant barley ( Hordeum vulgare L.). The same purification procedure was applied to adenylate kinases from leaves of maize ( Zea mays L.) and Chenopodium rubrum . In both these cases, isoforms of adenylate kinase could be purified using an additional affinity chromatography step. The main isoforms of adenylate kinase from all three plants exhibited apparent molecular masses between 25 and 31 kDa according to gel electrophoresis and gel filtration.

Adenylate kinase from plant tissues: Influence of ribonuclease on binding properties on Mono Q

Adenylate kinases modulate the three adenine nucleotide pools and were found to be localized as isoenzymes in different tissues and organelles in animals and plants. For investigations of adenylate kinase isoenzymes from plant tissues different plant extracts were examined by anion-exchange chromatography. During investigations with the strong anion exchanger Mono Q, adenylate kinase activity eluted in the void volume. This void volume activity did not always occur, but depended on the age of the plants and light treatment. The nature of the factors affecting void volume activity could only be partially resolved. It could be shown that RNase treatment at the beginning of extraction led to the disappearance of void volume activity, whereas an untreated extract still showed this activity.

Chlormadinone acetate suppresses prostaglandin biosynthesis in human endometrial explants

OBJECTIVE To elucidate the mode of action of chlormadinone acetate (CMA) in reducing dysmenorrheic pain by studying the effects of CMA and dexamethasone (DEX) on messenger RNA (mRNA) abundance of cyclo-oxygenase-2 (COX-2), annexin-1 (ANXA1), glucocorticoid receptor (GR), progesterone receptor (PR), and concentrations of prostaglandin F(2α) (PGF(2α)) and leukotrienes B(4) (LTB(4)) and C(4) (LTC(4)) in human endometrial explants. DESIGN Ex vivo study. SETTING University hospital. PATIENT(S) Fifteen premenopausal patients undergoing surgery for benign gynecologic disorders. INTERVENTION(S) Endometrial explants were obtained by aspiration curettage and stimulated ex vivo with interleukin-1β before exposure to CMA or DEX; mRNA levels were determined via reverse transcription-quantitative real-time polymerase chain reaction, and concentrations of arachidonic acid metabolites by enzyme immunoassays. MAIN OUTCOME MEASURE(S) Messenger RNA levels of COX-2, ANXA1, PR, and GR; concentrations of PGF(2α), LTB(4), and LTC(4) in endometrial explants treated with CMA or DEX. RESULT(S) In IL-1β-treated explants COX-2 mRNA and PGF(2α), concentrations were significantly down-regulated by CMA but not by DEX. Chlormadinone acetate did not affect mRNA abundance of ANXA1, PR, and GR. CONCLUSION(S) Our data suggest that CMA is a suppressor of COX-2 expression. Comparison with DEX revealed that progestin-specific activity of CMA may mainly be responsible for suppression of prostaglandin biosynthesis in human endometrium.